DNA ENCODING COBRA C3, CVF1, AND CVF2

Abstract: DNA sequences encoding cobra C3, CVF1, and CVF2, as well as plasmids and transformed hosts comprising such DNA sequences, are provided.

Description: Description DMA ENDODING COBRA C3. CVFl. AND CVF2 Technical Field The present invention relates to DNA encoding cobra C3, CVFl, and CVF2, plasmids comprising such DNA, and microorganisms transformed with such a plasmid. Background Art The third component of complement, C3, plays a pivotal role in both the classical and alternative pathways of complement activation, and many of the physiologic C3 activation products have important functions in the immune response and host defense (Mtiller-Eberhard, H.J. 1988. Molecular organization and function of the complement system. Annu . Rev. Biochem. 57:321). In the alternative pathway, the activated form of C3, C3b, is a structural subunit of the C3 convertase. This bimolecular enzyme consists of C3b and Bb, the activated form of factor B. This enzyme is formed by the binding of C3b to factor B that is subsequently cleaved by factor D, resulting in the formation of the C3 convertase, C3b,Bb, and the release of the activation peptide Ba. The C3 convertase activates C3 by cleaving the molecule into C3b and the anaphylatoxin, C3a. The C3b molecule will bind in close proximity to the C3 convertase. Eventually, the bound C3b will allow for the activation of C5 into C5b and the anaphylatoxin, C5a. C5 activation occurs by the same C3b,Bb enzyme that can cleave C5 when it is bound to an additional C3b molecule. The C5-cleaving enzyme is called C5 convertase. It is a tri olecular complex composed of (C3b) 2 ,Bb. Inasmuch as the activation of both C3 and C5 occurs at the identical active " site in the Bb subunit, the enzyme is also called C3/C5 convertase; and only one EC number has been assigned (EC 3.4.21.47). Cobra venom contains a structural and functional an¬ alog of C3 called Cobra Venom Factor (CVF) . This molecule can bind factor B in human and mammalian serum to form the complex, CVF,B (Hensley, P.M., C. O'Keefe, CJ. Spangler, J.C. Osborne, Jr.. and C.-W. Vogel. 1986. The effects of metal ions and temperature on the interaction of cobra venom factor and human complement factor B. J. Biol . Chem . 261:11038), which is also cleaved by factor D into the bimolecular enzyme CVF,Bb and Ba (Vogel. C.-W., and H.J. Mtiller-Eberhard. 1982. The cobra venom factor-dependent C3 convertase of human complement. A kinetic and thermodynamic analysis of a protease acting on its natural high molecular weight substrate. J. Biol . Chem . 257:8292). The bimolecular complex CVF,Bb is a C3/C5 convertase that activates C3 and C5 analogously to the C3/C5 convertase formed with C3b (Vogel, C.-W. 1991. Cobra venom factor, the complement-activating protein of cobra venom. In Handbook of Natural Toxins, Vol. 5, Reptile and Amphibian Venoms . A.T. Tu, ed. Marcel Dekker, New York, p. 147) . Although the two C3/C5 convertases C3b,Bb and CVF,Bb share the molecular architecture, the active site-bearing Bb subunit, and the substrate specificity, the two enzymes exhibit significant functional differences. The CVF,Bb enzyme is physicochemically far more stable than C3b,Bb (Vogel. C.-W., and H.J. Mtiller-Eberhard. 1982. The cobra venom factor-dependent C3 convertase of human complement. A kinetic and thermodynamic analysis of a protease acting on its natural high molecular weight substrate. J. Biol . Chem . 257:8292; Medicus, R.G., 0. Gotze, and H.J. Mtiller- Eberhard. 1976. Alternative pathway of complement: recruitment of precursor properdin by the labile C3/C5 convertase and the potentiation of the pathway. J. Exp. Med. 144:1076), it is resistant to inactivation by the regulatory proteins factors H and 1 (Lachmann, P.J. and A. Halbwachs. 1975. The influence of C3b inactivator (KAF) concentration on the ability of serum to support complement activation. Clin . Exp. Immunol . 21:109; Nagaki, K. , K. Iida, M. Okubo, and S. Inai. 1978. Reaction mechanisms of |8-1H globulin. Int . Arch . Allergy Appl . Immunol . 57:221), it exhibits different kinetic properties (Vogel. C.-W., and H.J. Mtiller-Eberhard. 1982. The cobra venom factor- dependent C3 convertase of human complement. A kinetic and thermodynamic analysis of a protease acting on its natural high molecular weight substrate. J. Biol . Chem . 257:8292; Pangburn, M.K., and H.J. Mtiller-Eberhard. 1986. The C3 convertase of the alternative pathway of human complement. Biochem J. 235:723), and it does not require additional C3b for C5 cleavage (Miyama, A., T. Kato, S. Horai, J. Yokoo, and S. Kashiba. 1975. Trypsin-activated complex of human Factor B with cobra venom factor (CVF) , cleaving C3 and C5 and generating a lyctic factor for unsensitized guinea pig erythrocytes. I. Generation of the activated complex. Biken J. 18:193; Von Zabern, I.B. Hinsch, H. Przyklenk, G. Schmidt, and W. Vogt. 1980. Comparison of Naja n. naja and Naja h. haje cobra venom factors: correlation between binding affinity for the fifth component of complement and mediation of its cleavage. Immunobi ology 157:499). CVF and mammalian C3 have been shown to exhibit several structural similarities including immunologic cross-reactivity (Alper, CA. and D. Balavitch. 1983. Cobra venom factor: evidence for its being altered cobra C3 (the third component of complement. Science 191:1275; Eggertsen, G.A. , U. Hell an, and J. Sjδquist. 1983. Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from Indian cobra (Naja naja) . J. Immunol . 131:1920; Vogel, C.-W., CA. Smith, and H.J. Mtiller-Eberhard. 1984. Cobra venom factor: structural homology with the third component of human complement. J. Immunol . 133:3235; Grier, A.H., M. Schultz, and C.-W. Vogel. 1987. Cobra venom factor and human C3 share carbohydrate antigenic determinants. J. Immunol . 139:1245), amino acid composition (Vogel, C.-W., CA. Smith, and H.J. Mtiller-Eberhard. 1984. Cobra venom factor: structural homology with the third component of human complement. J. Immunol . 133:3235; Vogel, C.-W., and M.K. Pangburn. 1985. An improved statistical method for quantitation of similarities of amino acid compositions reveals homologies among complement proteins. Complement 2:81), circular dichroism spectra, and secondary structure (Vogel, C.-W., CA. Smith, and H.J. Mtiller-Eberhard. 1984. Cobra venom factor: structural homology with the third component of human complement. J. Immunol . 133:3235), electron microscopic ultrastructure (Vogel, C.-W., CA. Smith, and H.J. Mtiller-Eberhard. 1984. Cobra venom factor: structural homology with the third component of human complement. J. Immunol . 133:3235; Smith, CA. , C.-W. Vogel, and H.J. Mtiller-Eberhard. 1982. Ultra-structure of cobra venom factor-dependent C3/C5 convertase and its zymogen. Factor B of human complement. J. Biol . Chem . 257:9879; Smith, CA. , C.-W. Vogel, and H.J. Mtiller- Eberhard. 1984. MHC class III products: an electron microscopic study of the C3 convertases of human complement. J. Exp. Med. 159:324), and amino-terminal amino acid sequence (Vogel, C.-W., CA. Smith, and H.J. Mtiller-Eberhard. 1984. Cobra venom factor: structural homology with the third component of human complement. J. Immunol . 133:3235; Lundwall, A., U. Hellman, G. Eggertsen, and J. Sjδquist. 1984. Chemical characterization of cyanogen bromide fragments from the /..-chain of human complement factor C3. FEBS Lett . 169:57). Nevertheless, significant structural differences exist between the two molecules. Whereas C3 is a two-chain molecule with an apparent molecular mass, dependent on the species, of 170 to 190 kDa (Eggertsen, G.A. , U. Hellman, and J. Sjδquist. 1983. Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from Indian cobra (Naja naja) . J. Immunol . 131:1920; DeBruijn, M.H.L., and G.H. Fey. 1985. Human complement component C3: cDNA coding sequence and derived primary structure. Proc. Natl . Acad. Sci . USA 82:708; Alsenz, J. , D. Avila, H.P. Huemer, I. Esparza, J.D. Becherer, T. Kinoshita, Y. Wang, S. Oppermann, and J.D. Lambris. 1992. Phylogeny of the third component of complement C3: analysis of the conservation of human CR1, CR2, H, and B binding sites, ConA binding sites, and thioester bond in the C3 from different species. Dev. Comp. Immunol . 16:63; Vogel, C- W. , and H.J. Mtiller-Eberhard. 1984. Biochemical characterization of the third component of the cobra complement system. Dev. Comp. Immunol . 8:239), CVF is a three-chain molecule with an apparent molecular mass of 136 kDa (Vogel, C.-W., and H.J. Mtiller-Eberhard. 1984. Cobra venom factor: improved method for purification and biochemical characterization. J. Immunol . Methods 73:203) that resembles C3c, one of the physiologic activation products of C3 (Vogel, C.-W. 1991. Cobra venom factor, the complement-activating protein of cobra venom. In Handbook of Natural Toxins , Vol. 5, Reptile and Amphibian Venoms . A.T. Tu, ed. Marcel Dekker, New York, p. 147; Vogel, C.-W., CA. Smith, and H.J. Mtiller-Eberhard. 1984. Cobra venom factor: structural homology with the third component of human complement. J. Immunol , 133:3235). Another significant structural difference between C3 and CVF lies in their glycosylation, CVF has a 7.4% (w/w) carbohydrate content consisting mainly of N-linked complex-type chains with unusual α-galactosyl residues at the non-reducing termini (Vogel, C.-W., and H.J. Mtiller- Eberhard. 1984. Cobra venom factor: improved method for purification and biochemical characterization. J. Immunol . Methods 73:203; Gowda, D.C, M. Schultz, R. Bredehorst, and C.-W. vogel. 1992. Structure of the major oligosaccharide of cobra venom factor. Mol . Immunol . 29:335). In contrast, human and rat C3 exhibit a lower extent of glycosylation with different structures of their oligosac¬ charide chains (Hase, S. , N. Kikuchi, T. Ikenaka, and K. Inoue. 1985. Structures of sugar chains of the third component of human complement. J. Biochem . 98:863; Hirani, S., J.D. La bris, and H.J. Mtiller-Eberhard. 1986. Structural analysis of the asparagine-linked oligosacchardies of human complement component C3. Biochem . J. 233:613; Miki, K. , S. Ogata, Y. Misumi, and Y. Ikehara. 1986. Carbohydrate structures of the third component of rat complement. Biochem J. 240:691). The multifunctionality of the C3 protein, which interacts specifically with more than 10 different plasma proteins or cell surface receptors, has spurred significant interest in a detailed structure/function analysis of the molecule. For some ligands of C3 the binding sites have been assigned to more or less defined regions of the C3 polypeptide including factor H (Ganu, V.S., and H.J. Mtiuller-Eberhard. 1985. Inhibition of factor B and Factor H binding to C3b by synthetic peptide corresponding to residues 749-789 of human C3. Complement 2:27), properdin (Daoudaki, M.E., J.D. Becherer, and J.D. La bris. 1988. A 3 -amino acid peptide of the third component of complement mediates properdin binding. J. Immunol . 140:1577; Farries, T.C., T. Seya, R.A. Harrison, and JP. Atkinson. 1990. Competition for binding sites on C3b by CR1, CR2, MCP, factor B, and factor H. Complement Inflamm . 7:30), factor B (Fishelson, Z. 1981. Complement C3: a molecular mosaic of binding sites. Mol . Immunol . 28:545), and the complement receptors CR1 (Becherer, J.D., and J.D. Lambris. 1988. Identification of the C3b receptor-binding domain in the third component of complement. J. Biol . Chem . 263:14586), CR2 (Lambris, J.D., V.S. Ganu, S. Hirani, and H.J. Mtiller-Eberhard. 1985. Mapping of the C3d receptor (CR2-binding site) and a neoantigenic site in the C3d domain of the third component C3 α-chain. Proc. Natl . Acad. Sci . USA 82:4235; Becherer, J.D., J. Alsenz, and J.D. Lambris. 1989. Molecular aspects of C3 interactions and structural/functional analysis of C3 from different species. Curr. Top. Microbiol . Immunol . 153:45), and CR3 (Becherer, J.D., J. Alsenz, and J.D. Lambris. 1989. Molecular aspects of C3 interactions and structural/functional analysis of C3 from different species. Curr. Top. Microbiol . Immunol . 153:45; Wright, S.D., P.A. Reddy, M.T.C Jong, and B. W. Erickson, 1987. C3bi receptor (complement receptor type 3) recognizes a region of complement protein C3 containing the sequence Arg-Gly-Asp. Proc. Natl . Acad. Sci . USA 84:1965; Taniguchi-Sidle, A., and D.E. Isenman. 1992. Mutagenesis of the Arg-Gly-Asp (RGD) triplet of human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11B/CD18). _7. Biol . Chem . 267:635). The elucidation of structural differences between C3 and CVF, two closely related molecules that share some properties (e.g., formation of a C3/C5 convertase) but differ in others (e.g., susceptibility to regulation by factors H and I) can be expected to help identify functionally important regions of the C3 molecule. The inventors have recently discovered that CVF actually exists in two forms, CVFl and CVF2. It is desirable to obtain large quantities of cobra C3, CVFl, and CVF2 for a number of reasons. However, the isolation of large quantities of the peptides from cobras is problematic to say the least. Thus, it is desirable to clone the genes which encode cobra C3, CVFl, and CVF2. Disclosure of the Invention Accordingly, one object of this invention is to provide a novel sequence of DNA which encodes cobra C3. It is another object of the present invention to provide a novel sequence of DNA which encodes CVFl. It is another object of the present invention to provide a novel sequence of DNA which encodes CVF2. It is another object of the present invention to provide a plasmid which comprises a sequence of DNA which encodes cobra C3. It is another object of the present invention to provide a plasmid which comprises a sequence of DNA which encodes CVFl. It is another object of the present invention to provide a plasmid which comprises a sequence of DNA which encodes CVF2. It is another object of the present invention to provide a microorganism which has been transformed with a plasmid comprising a sequence of DNA which encodes cobra C3. It is another object of the present invention to provide a microorganism which has been transformed with a plasmid comprising a sequence of DNA which encodes CVFl. It is another object of the present invention to provide a microorganism which has been transformed with a plasmid comprising a sequence of DNA which encodes CVF2. It is another object of the present invention to provide a method for producing large quantities of cobra C3. It is another object of the present invention to provide a method for producing large quantities of CVFl. It is another object of the present invention to provide a method for producing large quantities of CVF2. These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' cloning of the genes which encode cobra C3, CVFl, and CVF2. Brief Description of the Drawings A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein: Figure 1 depicts a map of clones used for the sequencing of cobra C3. The upper portion shows a schematic drawing of cobra C3 mRNA in which the positions and numbers of amino acid residues of the α- and ..-chains are indicated. The lower portion shows the relative positions of the five cDNA clones that were used to sequence the molecule; Figure 2 shows the cDNA and derived amino acid sequence of cobra C3. The NH 2 - and C-termini of the α- and /3-chains, functionally important regions, and known ligand binding sites are indicated. Amino acid residue numbering starts at the NH 2 -terminus of the pro-C3 molecule (NH 2 — terminus of the mature ,3-chain) ; Figure 3 illustrates dot plot comparisons of the cobra C3 protein sequence to those of human C3 (A) , rat C3 (B) , and urine C3 (C) . The dot plots were generated using a sequence analysis program (Devereux, J.R., P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). In that program, a moving window of 30 amino acid residues is compared, and where 21 or more residues are similar a dot is drawn on the graph. The arrow indicates the position of the α-chain/jS-chain junction; Figure 4 shows hydophilicity/hydrophobicity plots of cobra, human, and rat C3 proteins. The plots were generated using a sequence analysis program (Devereux, J.R., P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Hydrophilic regions are shown above, hydrophobic regions below the line. The locations of functionally important sites are indicated; Figure 5 provides a comparison of C3 sequences at the junction of the - and p " -chain. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and similarity with the cobra sequence is shown on the right. The C-terminus of the /3-chain and the NH 2 -terminus of the α-chain are indicated demonstrating the highly conserved sequence of four arginine residues in the pre-pro-C3 proteins in all species; Figure 6 provides a comparison of C3 sequences at the convertase cleavage site. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and similarity with the cobra sequence is shown on the right. The C3 convertase cleavage site is indicated; Figure 7 provides a comparison of C3 sequences at the thioester site. Comparisons were made with a sequence analysis program (Devereux, J.R., P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and simi¬ larity with the cobra sequence is shown on the right. The cysteine and glutamine residues involved in the intramolecular thioester are identified by asterisks; Figure 8 provides a comparison of C3 sequences at the factor B binding site. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and similarity with the cobra sequence is shown on the right; Figure 9 provides a comparison of C3 sequences at the factor H and CR2 binding sites. The upper panel shows the factor H orientation site. The lower panel shows the dis¬ continuous factor H binding site that includes the CR2 binding site (residues 1186-1197) with the highly conserved LYNVEA sequence in all mammalian C3 proteins. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and similarity with the cobra sequence is shown on the right; Figure 10 provides a comparison of C3 sequences at the properdin binding site. Comparisons were made with a sequence analysis program (Devereux, J.R., P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and similarity with the cobra sequence is shown on the right; Figure 11 provides a comparison of sequences required for active C3a peptide. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and similarity with the cobra sequence is shown on the right; Figure 12 provides a comparison of C3 sequences at the disputed CR3 binding site. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387) . Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the cobra C3 sequence as shown in Figure 2. The percent sequence identity and similarity with the cobra sequence is shown on the right; Figure 13 depicts a map of clones used for the sequencing of CVFl. The upper portion shows a schematic drawing of CVFl cDNA in which the positions and numbers of amino acid residues of the α-, γ-, and /3-chains are indicated. The lower portion shows the relative positions of the five cDNA clones that were used to sequence the molecule; Figure 14 shows the cDNA and derived amino acid sequence of CVFl. The NH 2 - and C-termini of the α-, 7-, and /3-chains, functionally important regions, and known ligand binding sites are indicated. Amino acid residue numbering starts at the NH 2 -terminus of the pro-CVFl molecule; Figure 15 provides a comparison of CVFl and C3 sequences at the factor B binding site. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the CVFl sequence as shown in Figure 14. The percent sequence identity and similarity with the CVFl sequence is shown on the right; Figure 16 provides a comparison of CVFl and C3 sequences at the properdin binding site. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the CVFl sequence as shown in Figure 14. The percent sequence identity and similarity with the CVFl sequence is shown on the right; Figure 17 provides a comparison of CVFl and C3 sequences at the thioester site. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the CVFl sequence as shown in Figure 14. The percent sequence identity and similarity with the CVFl sequence is shown on the right; Figure 18 provides a comparison of C3 sequences at the convertase cleavage site with the N-terminus of the 7-chain of CVFl. Comparisons were made with a sequence analysis program (Devereux, J.R., P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the CVFl sequence as shown in Figure 14. The percent sequence identity and similarity with the CVFl sequence is shown on the right; Figure 19 provides a comparison of CVFl and C3 sequences at the factor H and CR2 binding sites. The upper panel shows the factor H orientation site. The lower panel shows the discontinuous factor H binding site that includes the CR2 binding site (residues 1180-1191) with the highly conserved LYNVEA sequence in all mammalian C3 proteins. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the CVFl sequence as shown in Figure 14. The percent sequence identity and similarity with the cobra se¬ quence is shown on the right; Figure 20 shows hydophilicity/hydrophobicity plots of CVFl and cobra and human C3 proteins. The plots were generated using a sequence analysis program (Devereux, J.R., P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Hydrophilic regions are shown above, hydrophobic regions below the line. The locations of functionally important sites are indicated; Figure 21 provides a comparison of cobra, human, and mouse C3 with: (a) the N-terminal CVFl α-chain; (b) the N- terminal CVFl /S-chain; and (c) the N-terminal CVFl 7-chain. Comparisons were made with a sequence analysis program (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Identical amino acid residues are boxed and shaded whereas conservative replacements are shaded only. Amino acid residue numbering is based on the CVFl sequence as shown in Figure 14. The percent sequence identity and similarity with the cobra sequence is shown on the right; and Figure 22, shows the partial cDNA sequence of CVF2. Best Mode for Carrying out The Invention Thus, in a first embodiment, the present invention relates to DNA encoding cobra C3. Due to the occurrence of CVF in cobra venom, the complement system of cobra has received special attention. The existence of a complement system in cobra resembling the mammalian complement system in its molecular organization has been described (Vogel, C.-W., and H.J. Muller-Eberhard, 1985. The cobra complement system: I. The alternative pathway of activation, Dev. Comp. Immunol . 9:311; Vogel, C.W. , and H.J. Mtiller-Eberhard. 1985. The cobra complement system: II. The membrane attack complex. Dev. Comp. Immunol . 9:327). Cobra C3 was first purified from cobra plasma by affinity chromatography on an anti-CVF column, taking advantage of the cross-reaction of some antisera to CVF with cobra C3 (Eggertsen, G.A. , U. Hellman, and J. Sjδquist. 1983. Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from Indian cobra (Na a naja) . J. Immunol . 131:1920; Vogel, C.-W., and H.J. Miiiler-Eberhard. 1984. Biochemical characterization of the third component of the cobra complement system. Dev. Comp. Immunol . 8:239). More recently, cobra C3 was purified by conventional chromatography (Alsenz, J. , D. Avila, H.P. Huemer, I. Esparza, J.D. Becherer, T. Kinoshita, Y. Wang, S. Oppermann, and J.D. Lambris. 1992. Phylogeny of the third component of complement C3: analysis of the conservation of human CR1, CR2, H, and B binding sites, ConA binding sites, and thioester bond in the C3 from different species. Dev. Comp. Immunol . 16:63; Petrella, E.C., R. Bredehorεt, and C.-W. Vogel. 1989. Purification of cobra C3: initial characterization and comparison to cobra venom factor. Complement Inflamm . 6:386). The molecule has been shown to consist of an α- and jS-chain with apparent molecular masses of 106 to 112 kDa (o-chain) and 60 to 68 kDa (/S-chain) (Eggertsen, G.A. , U. Hellman, and J. Sjδquist. 1983. Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from Indian cobra (Naja naja) . J. Immunol . 131:1920; Alsenz, J., D. Avila, H.P. Huemer, I. Esparza, J.D. Becherer, T. Kinoshita, Y. Wang, S. Oppermann, and J.D. Lambris. 1992. Phylogeny of the third component of complement C3: analysis of the conservation of human CR1, CR2, H, and B binding sites, ConA binding sites, and thioester bond in the C3 from different species. Dev. Comp. Immunol . 16:63; Vogel, C- W. , and H.J. Mtiller-Eberhard. 1984. Biochemical characterization of the third component of the cobra complement system. Dev. Comp. Immunol . 8:239; Petrella, E.C, R. Bredehorst, and C.-W. Vogel. 1989. Purification of cobra C3: initial characterization and comparison to cobra venom factor. Complement Inflamm . 6:386). Cobra C3 has been shown to contain a thioester in its α-chain as is expected for C3 molecules (Alsenz, J., D. Avila, H.P. Huemer, I. Esparza, J.D. Becherer, T. Kinoshita, Y. Wang, S. Oppermann, and J.D. Lambris. 1992. Phylogeny of the third component of complement C3: analysis of the conservation of human CRl, CR2, H, and B binding sites, ConA binding sites, and thioester bond in the C3 from different species. Dev. Comp. Immunol . 16:63; Vogel, C- W., and H.J. Mtiller-Eberhard. 1984. Biochemical characterization of the third component of the cobra complement system. Dev. Comp. Immunol . 8:239). In contrast to all C3 molecules so far studied, cobra C3 is not glycosylated (Alsenz, J. , D. Avila, H.P. Huemer, I. Esparza, J.D. Becherer, T. Kinoshita, Y. Wang, S. Oppermann, and J.D. Lambris. 1992. Phylogeny of the third component of complement C3: analysis of the conservation of human CRl, CR2, H, and B binding sites, ConA binding sites, and thioester bond in the C3 from different species. Dev. Comp. Immunol . 16:63; Petrella, E.C, R. Bredehorεt, and C.-W. Vogel. 1989. Purification of cobra C3: initial characterization and comparison to cobra venom factor. Complement Inflam . 6:386). The present inventors have cloned and sequenced the cDNA for complement component C3 from cobra plasma. There are several lines of evidence that the sequence obtained is the sequence of cobra C3: l) the initial three cDNA clones were identified in an expression system using a mono- specific antiserum to cobra C3: 2) the derived NH 2 -terminal sequences of both α- and jS-chain match those of the amino acid sequences obtained by protein sequencing (Petrella, E.C, R. Bredehorεt, and C.-W. Vogel. 1989. Purification of cobra C3: initial characterization and comparison to cobra venom factor. Complement Inflamm . 6:386); 3) the derived amino acid compositions of cobra C3 and itε two chains resemble those obtained by protein hydrolysiε (Petrella, E.C, R. Bredehorεt, and C.-W. Vogel. 1989. Purification of cobra C3: initial characterization and comparison to cobra venom factor. Complement Inflamm . 6:386); and 4) the derived amino acid sequence shows a high degree of overall homology with that of C3 molecules from other species including highly conserved regions corresponding to known sites of functional importance. There are several lines of evidence that the obtained sequence representε that of cobra C3 and not of CVF: 1) CVF is not expected to be expressed in cobra liver; 2) the derived amino acid sequence reported here does not match the known stretches of protein sequence of CVF (Eggertsen, G., P. Lind, J. Sjδquist. 1981. Molecular characterization of the complement activating protein in the venom of the Indian cobra (Naja n . siamensis) . Mol . Immunol . 18:125); and 3) the cDNA sequence reported here differε from the cDNA sequence of CVF (Fritzinger, D.C, R. Bredehorεt, and C.W. Vogel. 1992. Complete sequence of two different cobra venom factor cDNAε. FASEB J. 6:A2998) . The primary structure of cobra C3 as derived from the sequencing of its cDNA shows a high degree of homology with those of C3 molecules from other species of which human (DeBruijn, M.H.L., and G.H. Fey. 1985. Human complement component C3: cDNA coding sequence and derived primary structure. Proc. Natl . Acad. Sci . USA 82:708), mouse (Lundwall, A., R.A. Wetsel, H. Domdey, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: I. Nucleotide sequence of cloned complementary and genomic DNA coding for the β-chain. J. Biol . Chem . 259:13851; Wetsel, R.A., A. Lundwall, F. Davidson, T. Gibson, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: II. Nucleotide sequence of cloned complementary DNA coding for the α-chain. J. Biol . Chem. 259:13857), and rat (Misu i, Y., M. Sohda, and Y. Ikehara. 1990. Nucleotide and deduced amino acid sequence of rat complement C3. Nucleic Acids Res . 18:2178) are fully and those of rabbit (Kusano, M. , N.H. Choi, M. Tomita, K.I. Mamamoto, S. Migita, T. Sekiya, and S. Niεhimura. 1986. Nucleotide sequence of cDNA and derived amino acid sequence of rabbit complement component C3 α-chain. Immunol . Inv. 15:365) and X. laevis (Grosεberger, D. , A. Marcuz, L. Du Paεquier, and J.D. Lambris. 1989. Conservation of structural and functional domainε in complement component C3 of Xenopus and mammalε. Proc. Natl . Acad. Sci . USA 86:1323) partially known. One intereεting difference is the absence of glycosylation sites consistent with a lack of detectable glycosylation of the protein. The overall εtructure of C3 molecules throughout the vertebrates seems to be highly conserved. As iε the caεe for mammalian C3, cobra C3 iε synthesized as a pre-pro molecule that is subεequently proceεsed into the mature two-chain protein by removing the signal peptide and the four arginine residues between the β- and α-chain. As with human C3, cobra C3 has a 22-amino acid εignal sequence and an α-chain of 992 amino acids. The /3-chain of cobra C3 with 633 residueε iε 12 residues shorter than the S-chain of human C3. All 27 cysteine residues are conserved in both molecules indi¬ cating a very high degree of εimilarity in the tertiary structure of the two proteins. With thiε degree of overall homology between cobra and mammalian C3ε, it iε not εurpriεing that several of the functional sites of C3 are highly conserved. These include the four arginine residues in the pre-pro-C3 molecule, probably required for proper maturation to yield the secreted C3 molecule, and the thioester εite, the unique structure in the α-chain required for alternative pathway activation and covalent binding to target cells. As in other C3 molecules, the glutamic acid reεidue whoεe 7-carboxyl group iε part of the intramolecular thioester with the cysteine residue is encoded as glutamine that must be converted into the thioester structure by a hitherto unknown process of post- translational modification. In addition, the C3 convertaεe cleavage εite and several binding sites for known ligands of C3 εuch as factor B, factor H, and properdin are highly conserved in cobra C3. The sequence homology at these four sites is εignificantly higher than the overall sequence homology throughout the molecule that strongly suggests that these sites are conεerved sites with defined functions. The observation that conεerved amino acid sequences are obεerved at functionally important sites where one or, in the case of the C3 convertase cleavage site, two protein ligands must interact with the molecule is not surprising: because ligands muεt evolve to match each other changeε in theεe binding sites are less likely to occur. The overall similarity of cobra C3 to C3s from other species is not surpriεing, given that the functional and molecular organization of the complement system has remained generally the same throughout the vertebrates. However, not all functionε of the mammalian complement system may be present in lower vertebrates, because the system may have undergone a functional diversification throughout phylogeny. Whereas the overall sequence homology of the C3a domain does not exceed the overall homology throughout the molecule, the last five amino acid residueε at the C-terminus of the C3a domain may suggeεt, that an active C3a anaphylatoxin is derived from cobra C3. However, in the case of the CR2 and CR3 binding siteε, no sequence homology exceeding that of the overall homology was found. Accordingly, the existence of CR2 and CR3 binding sites on cobra C3 and, therefore, the existence of these two cell surface ligands for C3 activation productε on cobra WBC cannot be deduced. However, indirect evidence for the existence of CR2 in reptiles may be derived from the observation, that human CR2 can bind to iC3 from Xenopus , a phylogenetically lower species (Alsenz, J., D. Avila, H.P. Huemer, I. Esparza, J.D. Becherer, T. Kinoshita, Y. Wang, S. Oppermann, and J.D. Lambris. 1992. Phylogeny of the third component of complement C3: analysis of the conservation of human CRl, CR2, H, and B binding siteε, ConA binding sites, and thioester bond in the C3 from different species. Dev. Comp. Immunol . 16:63). With regard to the CR3 binding site, recent work strongly suggests that the proposed region in the C-terminal portion of the C3 α-chain may not represent the CR3 binding εite altogether (Taniguchi-Sidle, A., and D.E. Isenman. 1992. Mutagenesis of the Arg-Gly-Asp (RGD) triplet of human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11B/CD18) . J. Biol . Chem . 267:635). The observed substitutionε in cobra C3 in the otherwiεe highly conεerved R(L)GD and DATMSI stretches may actually lend further support to that contention. In addition to being the first complement protein cloned from a reptile, cobra C3 also representε the firεt plasma protein cloned from cobra and the third protein from cobra for which cDNA sequence iε available. An interesting difference of cobra C3 to mammalian C3s iε the codon usage (see Table 1) . Whereas the G+C percentage of all known mammalian C3 mRNA is more than 53% (DeBruijn, M.H.L. , and G.H. Fey. 1985. Human complement component C3: cDNA coding sequence and derived primary structure. Proc. Natl . Acad. Sci . USA 82:708; Lundwall, A., R.A. Wetsel, H. Domdey, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: I. Nucleotide sequence of cloned complementary and genomic DNA coding for the β- chain. J. Biol . Chem. 2591:3851; Wetsel, R.A. , A. Lundwall, F. Davidson, T. Gibson, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: II. Nucleotide sequence of cloned complementary DNA coding for the α-chain. J. Biol . Chem . 2591:3857), the G+C percentage of cobra C3 mRNA is significantly lower at 43%. At this point, the significance of this difference is not known. However, it may reflect a different preference for codon usage in the species cobra because the G+C percentage of cobra nerve growth factor (43.6%) and of the cobra acetylcholine receptor (44.2%) resembles that of cobra C3 (Selby, M.J., R.H. Edwards, and W.J. Rutter. 1987. Cobra nerve growth factor; structure and evolutionary comparison. J. Neurosci . Res . 18:293; Neumann, D., D. Barchen, M. Horowitz, E. Kochva, and S. Fuchs. 1989. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteinε of the alpha-subunit. Proc. Natl . Acad. Sci . USA 86:7755). The DNA sequence of the present invention for cobra C3 comprises any DNA sequence that encodes the amino acid sequence of: a) pre-pro-cobra C3, which corresponds to the amino acid sequence of from poεition about -22 to poεition about 1629 in Figure 2; (b) pro-cobra C3, which corresponds to the amino acid sequence of from position about 1 to position about 1629 in Figure 2; (c) the α-chain of cobra C3, which corresponds to the amino acid sequence of from position about 638 to position about 1629 of Figure 2; (d) the /S-chain of cobra C3, which corresponds to the amino acid sequence of from position about 1 to position about 633 of Figure 2; (e) the α'-chain of cobra C3, which corresponds to the amino acid εequence of from poεition about 717 to poεition about 1629 in Figure 2; or (f) cobra C3a, which correεpondε to the amino acid sequence of from poεition about 638 to poεition about 716 in Figure 2. More preferably, the DNA εequence of the present invention for cobra C3 comprises: (a") the DNA sequence of from position about 1 to position about 5211 shown in Figure 2; (b') the DNA sequence of from position about 9 to position about 4961 in Figure 2; (c') the DNA sequence of from position about 75 to position about 4961 in Figure 2; (d') the DNA sequence of from position about 1986 to poεition about 4961 in Figure 2; (e') the DNA sequence of from about 75 to position about 1973 in Figure 2; (f) the DNA sequence of from position about 2223 to position about 4961 in Figure 2, or (g') the DNA sequence of from position about 1986 to position about 2222 in Figure 2. Thus, the DNA of the present invention for cobra C3 comprises a sequence which may encode pre-pro-cobra C3, pro-cobra C3, cobra C3, the α- chain of cobra C3, the j8-chain of cobra C3, the α'-chain of cobra C3, or cobra C3a. Of course, it iε to be underεtood that the present DNA sequences for cobra C3 encompass those which are derived from the DNA εequence shown in Figure 2 by making any number of additions, deletions, and/or εubεtitutions, so long as the polypeptide encoded possesses subεtantially the same properties as cobra C3. In another embodiment, the preεent invention provideε the DNA encoding CVFl. The cDNA sequence for CVFl is 5924 nucleotideε long, containing a εingle open reading frame of 4926 nucleotideε, coding for a εingle pre-pro protein of 1642 amino acid residues. The reported εequence has a 5' untranslated region of 3 nucleotides and a 3' untranslated sequence of 994 nucleotides. There are several lines of evidence that support the concluεion that the cDNA iε indeed the cDNA for CVFl. First of all, the derived protein sequences at the N- termini of α-, |8-, and 7-chains match those of the N- termini of the protein, with a single miεmatch in each εequence (data not shown) . Secondly, the location of glycosylation siteε iε similar to that found in the protein, with 2 or 3 εiteε found in the α-chain, a εingle εite in the /S-chain, and no sites found in the 7-chain. Finally, the similarity to the sequence of cobra C3 implies that we have sequenced a C3 related protein. Since the mRNA used for this sequence was isolated from the venom gland of cobras, and the sequence is different from that of cobra C3, it is likely that the sequence is that of CVFl. From the cDNA sequence, it is clear that CVFl, like cobra and other C3 proteins, is transcribed and translated as a single pre-pro-protein, which iε then processed to form the mature protein. In the case of CVFl, this processing includes the removal of the εignal sequence from the N-terminus of the α-chain, the removal of the 4 arginines and the H C3a" region that lie between the α- and 7-chains, the removal of the M C3d.g" region that lieε between the C-terminuε of the 7-chain and the N-terminuε of the /S-chain, and the glycosylation that occurs on the 2 (or 3) sites in the α-chain and the single εite that occurs in the /S-chain. While it is not known if all 3 εiteε in the α-chain are glycosylated, it εeems likely that the close proximity of the two siteε at positions 131 and 136 would not allow the glycosylation of one if the other is already glycosylated. As stated above, CVFl εhows a great deal of homology to cobra C3, both at the protein and at the nucleic acid level. One of the goals in sequencing both cobra C3 and CVFl was to determine if the two proteins are derived from the same gene (through differential processing at either the RNA or protein level) or from different genes. Comparing the CVF cDNA sequence to that of cobra C3 showε that the two proteinε are derived from different, though closely related genes. The main reason for this conclusion iε that the comparison of the two nucleic acid sequences εhowε that the εimilarity iε spread throughout the molecule, with differences not localized to discreet regions. If CVFl were a product of differential processing at the protein level, it would be expected that the cDNA sequences would be identical throughout. If the differential processing takes place at the RNA level, then one would expect to see portions of the sequence that are identical, interspersed with regions that have little or no similarity to one another. Since the two cDNAs are highly εi ilar throughout their lengths, it is most likely that they are derived from two different genes that are closely related to one another. The Thioester site and Factor H binding site of CVFl and cobra C3 are remarkably similar, even though neither is present in the mature CVFl protein. The degree of similarity found in this region, where there is no selective pressure to maintain the homology, is further proof that the CVFl gene arose quite recently. The similarity between the two genes iε also evident in the "C3a" region, that also is not present in the mature protein, and in the first 200 nucleotides of the 3' untranslated region, again implying that CVFl and C3 only recently diverged from one another. Recently, protease activitieε have been characterized in cobra venom that are able to cleave human C3 into a form that resembles C3b functionally, but has a similar subunit εtructure to CVFl (O'Keefe, M.C, L.H. Caporale, and C.W. Vogel. 1988. A novel cleavage product of human complement component C3 with εtructural and functional propertieε of cobra venom factor. J. Biol . Chem. 263:12690). Since thiε activity appears to be specific, and not just a random protease, it is possible that this protease serves in the maturation pathway of CVFl. Comparing the venom protease cleavage sites in human C3 to the procesεing εiteε in CVFl εhowε that the enzyme cleaveε human C3 at a position 11 amino acid residues downstream from the actual CVFl procesεing site at the N-terminus of the 7-chain, though the venom proteaεe site appears to be in the middle of one of the propoεed Factor B binding sites. The second venom protease cleavage site is in a position similar to the C— terminus of the 7-chain, though this position has not been mapped in CVFl. The third venom protease cleavage site is in poεition 71 amino acidε downstream from the N-terminus of the / S-chain. Given the complete structure of CVFl, and knowing the binding sites for certain regulatory proteins on C3, it should be possible to account for some of the unique properties of CVFl in activating complement. For example, it is known that, while Factors H and I are able to regulate the activation of complement by dissociating C3b,Bb (the C3 convertase), and by cleaving C3b, CVFl is reεiεtant to thiε regulation. Mapping the Factor H binding site on CVFl shows that the binding site is in the "C3d.g" domain that is removed during the maturation of the protein. Therefore, Factor H is unable to bind to the CVFl containing C3/C5 convertase, preventing Factor I from cleaving the CVF moiety of the convertase. It is also interesting to εpeculate on the intrinεic εtability of the CVFl containing C3/C5 convertaεe compared to the enzyme that contains C3. Comparing the Factor B binding sites to the two proteins εhould provide some insight into the increased εtability of the CVFl,Bb complex. One difference between the factor B binding εite iε the replacement of the serine at position 721 of CVFl with an acidic amino acid in C3s. The DNA εequence of the preεent invention for CVFl comprises any DNA sequence encoding a polypeptide having the amino acid sequence of: (g) pre-pro-CVFl, which corresponds to the amino acid sequence of from position about -22 to position about 1620 in Figure 14; (h) pro- CVF1, which corresponds to the amino acid sequence of from position about 1 to position about 1620 in Figure 14; (i) the amino acid sequence of from position about 1 to position about 1010 in Figure 14; (j) the amino acid sequence of from position about 711 to poεition about 1620 in Figure 14; (k) the α-chain of CVFl, which corresponds to the amino acid sequence of from position about 1 to position about 627 in Figure 14; (1) the 7-chain of CVFl, which correspondε to the amino acid sequence of from position about 711 to position about 1010 in Figure 14; or (m) the S-chain of CVFl, which corresponds to the amino acid sequence of from position about 1242 to position about 1620 in Figure 2. More preferably, the DNA sequence of the present invention for CVFl compriεeε: (h') the DNA sequence of from position about 1 to position about 5929 in Figure 14; (i') the DNA sequence of from position about 4 to position about 4929 in Figure 14; (j') the DNA sequence of from position about 70 to position about 4929 in Figure 14; (k') the DNA sequence of from position about 70 to position about 3299 in Figure 14; (1') the DNA εequence of from position about 2200 to poεition about 4929 in Figure 14; (m') the DNA εequence from poεition about 70 to poεition about 951 in Figure 14; (n') the DNA εequence of from position about 2200 to position about 3299 in Figure 14; or (o') the DNA sequence of from position about 3793 to position about 4929 in Figure 14. Thus, the DNA of the present invention for CVFl may comprise a DNA sequence which encodes pre-pro-CVFl, pro- CVF1, the α-chain of CVFl, the /S-chain of CVFl, the portion of pro-CVFl which contains from the amino-terminus of the α-chain to the carboxy-terminuε of the 7-chain; or the portion of pro-CVFl which contains from the amino-terminus of the 7-chain to the carboxy-terminus of the /3-chain. Of courεe, it should be understood that the present DNA sequence encoding CVFl encompasses those derived from the sequence shown in Figure 14 by any number of additions, deletions and/or substitutions, so long aε the encoded polypeptide possesses substantially the same properties aε CVFl, such as reεiεtance to Factor H and/or I control and the ability to form a convertaεe with b, which iε εtable and does not decay rapidly. In another embodiment, the present invention provides DNA sequences for CVF2 and derivatives thereof. A partial εequence encoding of CVF2 iε shown in Figure 22. A full length sequence encoding pre-pro-CVF2 may be constructed by ligating the 3' end of any DNA sequence which encodes for a polypeptide having the amino acid sequence of from position about -22 to position about 299, as shown in Figure 14 to the 5' end of any DNA sequence encoding a polypeptide having the amino acid sequence as shown in Figure 22. As in the case of CVFl, the DNA of the present invention for CVF2 may comprise any DNA sequence that encodes: (n) pre-pro-CVF2 , corresponding to the amino acid sequence in which the carboxy-terminus of the amino acid sequence of from position about -22 to position about 299 in Figure 14 iε bonded to the amino-terminus of the amino acid sequence of from poεition about 1 to poεition about 1333 in Figure 22; (o) pro-CVF2, corresponding to the amino acid sequence in which the carboxy-terminus of the amino acid sequence of from position about 1 to position about 299 in Figure 14 is bonded to the amino-terminus of the amino acid sequence of from position about 1 to position about 1333 in Figure 22; (p) the amino acid sequence in which the carboxy-terminus of the amino acid sequence of from position about 1 to position about 299 in Figure 14 iε bonded to the amino-terminus of the amino acid sequence of from position about 1 to position about 2147 in Figure 22; (q) from position about 1248 to position about 4001 in Figure 22; (r) the α-chain of CVF2, which corresponds to the amino acid sequence in which the carboxy terminus of the amino acid εequence from poεition about 1 to poεition about 299 in Figure 14 iε bonded to the amino acid εequence from position about 1 to position about 332 in Figure 22; (ε) the 7-chain of CVF2, which correεponds to the amino acid sequence from position about 416 to position about 715 in Figure 22; or (t) the /8-chain of CVF2, which corresponds to the amino acid sequence of from position about 947 to position about 1333 in Figure 22. More preferably, the DNA sequence for CVF2 comprises a DNA sequence corresponding to the sequence derived by ligating the 3' end of DNA sequence, X, from Figure 14 to the 5' end of DNA sequence, Y, from Figure 22, where X and Y are shown in the Table below: .£___ __ <P') 1 to 964 1 to 4138 (q') 4 to 964 1 to 4001 (r') 70 to 964 1 to 4001 (s') 70 to 964 1 to 2147 (f ) 70 to 964 1 to 998 (u') the DNA sequence from position about 1248 to position about 2147 in Figure 22; (v') the DNA sequence from position about 2841 to position about 4001 in Figure 22; or (W) the DNA sequence from position about 1248 to position about 4001 in Figure 22. Again, it is to be understood that the present sequence encoding CVF2 includes thoεe derived from the sequences shown in Figure(ε) 14 and/or 22 by any number of additions, deletions, and/or substitutions, so long as the encoded polypeptide possesses subεtantially the same properties as CVFl. In another embodiment, the present invention provides plasmids which comprise a DNA sequence encoding pre-pro- cobra C3, pro-cobra C3, the α-chain of cobra C3, the /S- chain of cobra C3, the α'-chain of cobra C3, Cobra C3a, pre-pro CVFl, pro-CVFl, the amino acid sequence of from position about 1 to position about 1010 in Figure 14, the amino acid sequence of from position about 711 to poεition about 1620 in Figure 14, the α-chain of CVFl, the 7-chain of CVFl, the /3-chain of CVFl, pre-pro CVF2, pro-CVF2, the amino acid εequence in which the carboxy-terminus of the amino acid sequence of from position about 1 to position about 299 in Figure 14 is bonded to the amino-terminus of the amino acid sequence of from position about 1 to position about 715 in Figure 22, the amino acid sequence from poεition about 416 to poεition about 1333 in Figure 22, the α-chain of CVF2, the 7-chain of CVF2, or the /S- chain of CVF2. Any plasmid suitable for cloning or expression may be uεed and the DNA may be inserted in the plasmid by conventional techniques. Suitable plasmids and the techniques uεed to inεert the DNA of the preεent invention into εuch plaεmids are well known to those skilled in the art. For expression purposes, the DNA should be inserted downstream from a promoter and in the proper reading frame. In another embodiment, the present invention provides transformed hosts which contain a DNA sequence encoding pre-pro-cobra C3, pro-cobra C3, the α-chain of cobra C3, the /3-chain of cobra C3, the α'-chain of cobra C3, cobra C3a, pre-pro CVFl, pro-CVFl, the amino acid sequence of from position about 1 to poεition about 1010 in Figure 14, the amino acid sequence of from position about 711 to position about 1620 in Figure 14, the α-chain of CVFl, the 7-chain of CVFl, the /3-chain of CVFl, pre-pro CVF2, pro- CVF2, the amino acid εequence in which the carboxy-terminus of the amino acid εequence of from poεition about 1 to poεition about 299 in Figure 14 iε bonded to the amino- terminus of the amino acid εequence of from position about 1 to position about 715 in Figure 22, the amino acid sequence from position about 416 to position about 1333 in Figure 22, the α-chain of CVF2, the 7-chain of CVF2, or the /3-chain of CVF2. Again, suitable hosts and the means for transfor ing them are well know to those skilled in the art. Examples of suitable prokaryotic hosts include: E coli , B . subtilis, etc. In the present case, it may be desirable to express the present genes in eukaryotic hostε such as CHO or NIH 3T3 cellε. In yet another embodiment, the present invention provides a method for preparing a polypeptide by culturing a transformed host comprising a DNA sequence encoding pre- pro-cobra C3, pro-cobra C3, the α-chain of cobra C3, the β- chain of cobra C3, the α'-chain of cobra 3, cobra C3a, pre- pro CVFl, pro-CVFl, the amino acid sequence of from position about 1 to position about 1010 in Figure 14, the amino acid sequence of from position about 711 to position about 1620 in Figure 14, the α-chain of CVFl, the 7-chain of CVFl, the /3-chain of CVFl, pre-pro CVF2, pro-CVF2, the amino acid sequence in which the carboxy-terminus of the amino acid sequence of from position about 1 to position about 299 in Figure 14 is bonded to the amino-terminus of the amino acid sequence of from position about 1 to position about 715 in Figure 22, the amino acid sequence of from position about 416 to position about 1333 in Figure 22, the α-chain of CVF2, the 7-chain of CVF2, or the β- chain of CVF2. The exact conditions required for the culturing will depend of course on the identity of the transformed host. However, selection of culture conditions is well within the abilities of the skilled artisan. It should be noted that although CVFl and CVF2 are glycosylated as naturally occurring, it has been discovered that these polypeptideε retain their activity even in the unglycosylated state. Thus, an active product may be obtained even if produced by a host incapable of effecting the proper glycosylation. Further, C3, CVFl, and CVF2 may be procesεed from the pre-pro-form by treatment with either whole cobra venom or the purified proteaεes from cobra venom, as described in the Doctoral thesiε of M. Clare O'Keefe, Georgetown Univerεity, 1991. Thuε, active C3, CVFl, and CVF2 may be obtained even when produced by a hoεt incapable of the proper post-translational processing. The CVFl and CVF2 produced by the present proceεε may be uεeful for the treatment of cancer. Thuε, CVFl or CVF2 may be bound to an antibody which recognizeε a tumor cell. In thiε way, the CVFl or CVF2 will be directed to the target cancer cells. Since, CVFl and CVF2 are insenεitive to factor H control, thiε method will lead to the εelective deεtruction of the cancer cellε. Likewise, cobra C3 may not be inactivated by human factor H and, thus, may be useful in the εame method. Other featureε of the invention will become apparent in the courεe of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof. EXAMPLES !__ Cobra C3: Materialε and Methods Materials. Solutions for RNA isolation, cDNA preparation, λgtll cloning, and hybridization probe labeling were obtained from Amersham (Skokie, IL) . Restriction enzymes were from either Pharmacia Fine Che icalε (Piscataway, NJ) or from New England Biolabs (Beverley, MA) . Plaεmid pUC 18 waε purchased from Boehringer Mannheim (Indianapolis, IN), and M13 mp 18 and mp 19 were purchased from New England Biolabs (Yanisch-Perron, C,J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mpl8 and pUC19 vectors. Gene 33:103). DNA modification enzymes were obtained from Pharmacia or New England Biolabs, and DNA sequencing reagents were obtained from United States Biochemicals (Cleveland, OH) . Anti-mouse IgG+IgM (alkaline phoεphataεe conjugated) was purchased from Tago (Burlingame, CA) . [α- 32 P]dATP, [α- 32 P]dCTP, and [α- 35 S]dATP were obtained from Amersham. RNA isolation from cobra liver. Adult cobras (Naja naja kaouthia , 1.5-2 meters in length) were premedicated with ketamine (-70 mg/kg i.m.) and anesthetized with halothane/oxygen after tracheal intubation essentially as deεcribed (Vogel, C.-W., and H.J. Muller-Eberhard, 1985. The cobra complement system: I. The alternative pathway of activation, Dev. Comp. Immunol . 9:311). The livers were removed and immediately frozen in liquid nitrogen. For RNA preparation, approximately one gram of tissue was εuεpended (while frozen) in 20 ml of a solution of 4 M guanidinium thiocyanate and 1.14 M /S-mercaptoethanol , and the RNA extracted according to the instructions supplied with the Amersham RNA Extraction Kit. This procedure iε based on a published procedure (Han, J.H., C. Stratowa, and W.J. Rutter, 1987. Isolation of full-length putative rat lyεophospholipase cDNA using improved methodε for mRNA iεolation and cDNA cloning. Biochemistry 26:1617). Poly-A containing RNA waε then isolated by chromatography over oligo-dT cellulose (Jacobson, A. Purification and fractionation of poly(A)* RNA. 1987. In Methods in Enzymology, Vol. 152. S.L. Berger and A.R. Kimmel, edε. Academic Press, Orlando, FL, p. 254) . cDNA synthesis and cloning. First εtrand (Krug, M.S., and S.L. Berger. 1987. Firεt-strand cDNA synthesis primed with oligo(dT). In Methods Enzymology. Vol. 152. S.L. Berger and A.R. Kimmel, eds. Academic Press. Orlando, FL, p. 316) and second strand (Gubler, U. 1987. Second-strand cDNA synthesis: mRNA fragments as a primer. In Methods in Enzymology, vol. 152. S.L. Berger and A.R. Kimmel. eds. Academic Press, Orlando. FL. p. 330) synthesiε of cobra liver cDNA was performed using the cDNA synthesis kit from Amersham. cDNA was synthesized using both oligo-dT and random hexa ers as the primerε. cDNA waε then prepared for cloning into λgtll (Wu. R. , T. Wu. and A. Ray. 1987. Adaptors, linkers, and methylation. In Methods in Enzymology. vol. 152. S.L. Berger and A.R. Kimmel, edε. Academic Preεε, Orlando, FL. p. 343) , ligated with λgtll armε, and the recombinant λ cloneε were packaged (Hohn, B. , and K. Murray. 1977. Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc. Natl . Acad. Sci . USA 74:3259). Escherichia coll Y1090(r " , m * ) was used as the hoεt for recombinant λgtll. Purification of cobra C3 and raiεino of mouse antisera to cobra C3. Cobra C3 was purified (Petrella, E.C, R. Bredehorεt, and C.-W. Vogel. 1989. Purification of cobra C3: initial characterization and comparison to cobra venom factor. Complement Inflamm. 6:386) and polyclonal antisera were raised in mice aε described (Grier, A.H. , M. Schultz, and C.-W. Vogel. 1987. Cobra venom factor and human C3 share carbohydrate antigenic determinants. J . Immunol . 139:1245) . Screening of λot 11 librarieε. Librarieε were εcreened with antibody probes after transferring recombinant proteins to nitrocellulose filters (Young, R.A. , and R.W. Davis. 1983. Yeaεt RNA polymerase II genes: isolation with antibody probes. Science 222:778; Huynh, T.V., R.A. Young, and R.W. Davis. 1985, Construction and screening cDNA libraries in λgtlO and λgtll. In DNA Cloning vol. 1. A Practical Approach. D.M. Glover, ed. IRL Press, Oxford, p 49) . Mouse anti-cobra C3 waε uεed aε the primary antibody for screening the liver library. Further plaque purification was done with εuccessively lower plaque densitieε. Later εcreening, using cDNA clones derived by the antibody screening procedure as probes, was done by hybridization (Wahl, G.M. , and S.L. Berger. 1987. Screening colonies of plaques with radioactive nucleic acid probes. In Methods in Enzymology, vol. 152. S.L. Berger and A.R. Kimmel, eds. Academic Press, Orlando, FL, p. 415) . These probes were labeled with [α- 32 P]dATP or [α- 3 P]dCTP (Feinberg, A.P., and B. Vogelstein. 1983. A technique for radio-labeling DNA reεtriction endonuclease fragments to high specific activity. Anal . Biochem . 132:6). Subcloning and DNA εeσuence analvεis. Clones containing C3 inserts were grown up on agarose plates and their DNA prepared as described (Maniatis, T. , E.F. Fritεch, and J. Sambrook. 1982. Molecular Cloning - A Laboratory Manual . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Inεerts were isolated by EcoRI digestion, followed by agarose gel electrophoresis. Fragments were eluted from the gel using an electroelution device (IBI, New Haven. CT) . The DNA insertε were then ligated into pUC 18 so that large quantities of the insert could be prepared for restriction analysis and DNA sequencing. Subfragments of the C3 inserts for sequencing were generated by digestion with restriction enzymes that cut at a 4 bp recognition sequence (Haelll, Hinfl, and Rsal) . These subfragments were then cloned into Smal cut M 13 mpl8 or mpl9 to generate sequencing templates. Sequencing was performed using the dideoxy-chain ter¬ mination technique (Sanger, F. S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain terminating inhibitors, Proc. Natl . Acad. Sci . USA 74:5463). The Sequenase Version 2.0 sequencing kit from U.S. Biochemicals was used as the source of enzymes, chemicals, and primers for sequencing (Tabor, S., and CC. Richardson. 1989. Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA Polymerase I. Proc. Natl . Acad. Sci . USA 86:4076). Sequences were analyzed using the Genetics Computer Group of the Wisconsin Biotechnology Center package of sequence analysis programs (Devereux, J.R. , P. Haeberli, and O.A. Smithies. 1984. Comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res . 12:387). Both strands were sequenced for approximately 85% of the reported sequence. For those regions where only one strand was sequenced, several nonambiguous gels were used to generate the consensus sequence. Sequence alignments were done with the gap program (Needleman, S.B., and CD. Wunsch. 1970. A general method applicable to the search for similarities in the amino acid sequences of two proteins. J. Mol . Biol . 48:443). Dot plot comparisons (Maizel, Jr., J.V. , and R.P. Lenk. 1981. Enhanced graphic matrix analyεiε of nucleic acid and protein εequenceε. Proc. Natl . Acad. Sci . USA 78:7665) and the determination of conεervative replacements of amino acids (Dayhoff, M.O., ed. 1978, Atlas of Protein Sequence and Structure , vol. 5, National Biomedical Research Foundation, Washington, DC) were performed as described. Hydrophilicity/ hydrophobicity plotε were generated as described (Hop, T.P., and K.R. Woods. 1981. Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl . Acad. Sci . , USA 78:3824). Sequence analyseε were performed with the reported εequenceε for C3 from human (DeBruijn, M.H.L., and G.H. Fey. 1985. Human complement component C3: cDNA coding εequence and derived primary structure. Proc. Natl . Acad. Sci . USA 82:708), mouse (Lundwall, A., R.A. Wetsel, H. Do dey, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: I. Nucleotide sequence of cloned complementary and genomic DNA coding for the β-chain J. Biol . Chem . 259:13851; Wetsel, R.A. , A. Lundwall, F. Davidson, T. Gibson, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: II. Nucleotide sequence of cloned complementary DNA coding for the α-chain. J. Biol . Chem . 259:13857), rat (Miεumi, Y., M. Sohda, and Y. Ikehara. 1990. Nucleotide and deduced amino acid sequence of rat complement C3. Nucleic Acids Res . 18:2178), rabbit (Kusano, M. , N.H. Choi, M. Tomita, K.I. Mamamoto, S. Migita, T. Sekiya, and S. Niεhimura. 1986. Nucleotide sequence of cDNA and derived amino acid sequence of rabbit complement component C3 α-chain. Immunol. Inv. 15:365), and Xenopus (Groεεberger, D. , A. Marcuz, L. Du Paεquier, and J.D. Lambris. 1989. Conservation of structural and functional domains in complement component C3 of Xenopus and mammals. Proc . Natl . Acad. Sci . USA 86:1323). The numbering of amino acid positions throughout the paper iε baεed on the cobra pro-C3 molecule, εtarting with number one at the NH 2 -terminuε of the /3-chain (comp. Fig. 2) . RESULTS Screening of the cobra liver library. Poly-A * RNA from cobra liver waε used for the preparation of cDNA, which was cloned into the EcoRI site of λgtll. Two libraries were prepared, one from cDNA that waε primed by oligo-dT, and one that was primed from a random mixture of oligonucleotides. Each library contained more than 5 x IO 6 cloneε. Initially, 5 x IO 5 plaques from the random primed library were screened for clones expressing cobra C3 by detection with the mouse anti-cobra C3 antisera. In our original screening, three cobra C3 clones (called C3-5, C3-6, and C3-7; 2.5, 2.4, and 1.6 kb, reεpectively; Fig. 1) were iεolated. Sequence analysis of the three C3 clones revealed that they all overlapped one another, containing a single open reading frame of 3709 bp. When the derived amino acid sequence of this protein was compared to that of human C3, it was found that the portion of cobra C3 that had been sequenced contained almost the entire S-chain and the NH j -terminal two-thirds of the α-chain, repreεenting approximately 75% of the mature protein. To iεolate clones representing the rest of the C3 mRNA, the liver library was rescreened by hybridization, using fragments of the original three clones as hybridization probes. Ten cloneε repreεenting the 3' end of the mRNA were isolated from the oligo-dT primed λgtll library, varying in size from 2.1 kb to almost 4 kb in length. The smallest of these, C3-37, was chosen for sequence analysis. Clones presumably repreεenting the 5' end of the C3 message were isolated from the random oligo primed library. Of these, one clone (C3-72: 0.9 kb) was sequenced. Figure 1 showε the placement of the five cloneε uεed for sequencing the cobra C3 mesεage. Structure of cobra C3 mRNA. The cobra C3 mRNA iε 5211 bp in length. It contains a single open reading frame of 4953 bp (Fig. 2). The cDNA has a 3' untranslated region of 250 bp, including a poly-A tail of seven residues. The translated mesεage codes for a εingle pre-pro-C3 molecule of 1651 amino acids εtarting at position 9 of the mRNA. The pre-pro-protein has a 22 amino acid residue signal sequence with a core rich in hydrophobic amino acidε. The εignal εequence is followed by the /8-chain of 633 amino acid residueε. The C-terminuε of the /8-chain iε separated from the NH 2 -terminus of the α-chain by four arginine reεidueε. The α-chain iε 992 amino acidε in length. There are no N-glycoεylation εites in either the α-chain or the /8-chain of cobra C3. Cobra C3 contains one N-glycoεylation recognition εequence Aεn-X-Ser at position 157-159. However, the residue at position 158 is proline, which haε been εhown to prevent glycoεylation (Bauεe, E. 1983. Structural requirements of N-glycosylation of proteins: εtudieε with proline peptideε aε conformational probes. Biochem . J. 209:331). The G+C percentage of the cobra C3 mRNA i-S 43%, compared to a G+C percentage of greater than 53% for mammalian C3 mRNA (DeBruijn, M.H.L. , and G.H. Fey. 1985. Human complement component C3: cDNA coding sequence and derived primary structure. Proc. Natl . Acad. Sci . USA 82:708; Lundwall, A., R.A. Wetsel, H. Domdey, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: I. Nucleotide sequence of cloned complementary and genomic DNA coding for the β-chain J. Biol . Chem . 259:13851; Wetεel, R.A., A. Lundwall, F. Davidson, T. Gibson, B.F. Tack, and G.H. Fey. 1984. Structure of murine complement component C3: II. Nucleotide sequence of cloned complementary DNA coding for the α-chain. J. Biol . Chem . 259:13857). The lower G+C content of cobra C3 is reflected in the codon usage, shown in Table I, where G+C-rich codons are underrepreεented aε compared to mammalian C3 genes. Table II gives the amino acid composition of the α-chain, the /S-chain, and the mature protein. The mature protein is made up of 1625 amino acid reεidueε and haε a molecular mass of 182 kDa consiεting of an α-chain of 112 kDa and a /3-chain of 70 kDa. Homolocrv of cobra C3 with C3 from other species. The cDNA sequence and the derived protein sequence of cobra C3 were compared with the corresponding εequenceε of C3 from other εpecieε for which the full (human, mouse, rat) or partial (rabbit, Xenopus laevis) sequences have been reported. As iε evident from Table III, the cDNA εequence of cobra C3 shows approximately 58% identity to the cDNA sequences of all five species. At the protein level, cobra C3 and the three fully sequenced mammalian C3s exhibit a sequence identity of approximately 52% and a sequence similarity of approximately 71% if one allows for conservative εubεtitutionε. Protein εequence identity and similarity are 2 to 3% lower for the partially sequenced portions of C3 from rabbit and Xenopus. Dot plot comparisons of the protein sequence of cobra C3 with those of three mammalian C3s indicate that there are large stretches of homology between the C3 proteins, with the homology extending throughout the molecule (Fig. 3). Similar results were obtained with dot plot comparisons of the nucleotide sequences (not shown) . The homology of cobra C3 with mammalian C3s is further evident by comparing the hydrophilicity/hydrophobicity plots as calculated from the amino acid sequences (Fig. 4) . A careful analysis of the plots indicates that the pattern of hydrophobic and hydrophilic regions is very similar -throughout all three C3 proteins. In particular, all three plots show the hydrophobic signal sequence, the predominantly hydrophilic C3a region, a short hydrophilic peak at the NH 2 -terminus of the α'-chain containing the factor H and B binding sites, a hydrophobic twin peak containing the thioester site, a relatively wide hydrophilic peak leading into a shorter hydrophobic peak at the factor H and CR2 binding site, and a wide hydrophilic peak followed by a small hydrophobic peak at the C-terminus. Proper aεεignment of the properdin binding εite and the diεputed CR3 binding site is difficult although the general pattern of the plotε in that region iε alεo similar. Cobra C3 contains 27 cyεteine reεidueε, of which 24 are in the α-chain and only three in the /3-chain. The total number and the distribution of the cysteines are identical to those in other C3 specieε, indicating that they are highly conserved. Other highly conserved εiteε include the polypeptide εtretch in the pre-pro-C3 molecule linking the C-terminuε of the /3-chain with the NH 2 -terminuε of the α-chain including the four arginine reεidueε that are being removed in the maturation process (Fig. 5) , the C3 convertase cleavage εite (Fig. 6) , the thioeεter site (Fig. 7) , and the factor B binding site (Fig. 8) . Cobra C3 also seems to have homologous binding siteε for factor H (Fig. 9) and properdin (Fig. 10) as well as a conserved sequence in the functionally important region of the C3a anaphylatoxin (Fig. 11). For human C3, the binding of factor H has been propoεed to occur at an "orientation εite" between residues 727 and 767 and a "discontinuous binding site" between residues 1187 and 1249 (human C3 numbering), both in the α-chain (Ganu, V.S., and H.J. Mtiuller-Eberhard. 1985. Inhibition of factor B and Factor H binding to C3b by synthetic peptide correεponding to residues 749-789 of human C3. Complement 2:27). At the orientation site the homology certainly exceeds the overall homology between cobra C3 and mammalian C3. At the discontinuous binding site, however, the homology does not exceed that of the overall homology of the two proteins. However, there are at least four highly conserved stretcheε within the factor H binding site. Collectively, these data strongly suggest that homologous binding sites exist for factor H in cobra C3. The homology at the properdin binding εite in the C- terminal portion of the α-chain iε alεo εignificantly above the overall homology, similarly suggesting a conserved binding site for properdin. The 21 C-terminal amino acid residueε of C3a, known to be able to elicit nearly 100% of C3a activity, εhow a degree of homology that doeε not exceed the overall ho- mology between cobra and mammalian C3s. However, four of the last five amino acid reεidueε including the C-terminal arginine, are identical and εimilarly suggest conservation of thiε region of the C3 molecule. More ambiguous is the homology of the CR2 binding site (Fig. 9) . Whereas all mammalian C3s show the highly conserved LYNVEA sequence within the CR2 binding site, cobra C3 only shares three of these six residues and shows an overall homology to mammalian C3s at the CR2 binding site that does not exceed the overall homology of the molecules. Accordingly, although cobra C3 may have a binding site for CR2, its existence cannot be deduced from sequence conservation by comparison with C3 from other species. Figure 12 shows the sequence comparison at the disputed CR3 binding site. Cobra C3 does not contain the RGD or LGD triplet but a still similar LGE triplet. However, the neighboring DATMSI sequence, which is highly conserved in other C3ε, shows two substitutions in cobra C3. In addition, the overall homology in this region between cobra C3 and the C3s from other species does not exceed the overall homology of the molecules. The existence of a CR3 binding site in cobra C3 in this region cannot be deduced from these data; and the difference in the RGD and DATMSI sequences may actually support the hypothesis that this particular region of C3 does not represent the CR3 binding site in any species (Taniguchi-Sidle, A., and D.E. Iεenman. 1992. Mutagenesis of the Arg-Gly-Asp (RGD) triplet of human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type III (CR3, CD11B/CD18). J. Biol . Chem . 267:635). TABLE I Codon frequency for cobra C3 and human C3 coding sequences' 11 Amino Cobra Human Amino Cobra Human Acid Codon C3 C3 Acid Codon C3 C3 Gly GGG 0.20 0.27 Trp UGG 1.00 1.00 GLY GGA 0.37 0.17 End UGA 0.00 0.00 Gly GGU 0.21 0.12 Cys UGU 0.56 0.31 Gly GGC 0.23 0.44 Cys UGC 0.44 0.69 Glu GAG 0.33 0.69 End UAG 0.00 0.00 Glu GAA 0.67 0.31 End UAA 0.00 0.00 Asp GAU 0.69 0.28 Tyr UAU 0.57 0.16 Asp GAC 0.31 0.72 Tyr UAC 0.43 0.82 Val GUG 0.42 0.50 Leu UUG 0.20 0.10 Val GUA 0.14 0.05 Leu UUA 0.11 0.01 Val GUU 0.22 0.12 Phe UUU 0.59 0.22 Val GUC 0.22 0.33 Phe UUC 0.41 0.78 Ala GCG 0.03 0.08 Ser UCG 0.03 0.10 Ala GCA 0.37 0.14 Ser UCA 0.18 0.08 Ala GCU 0.38 0.19 Ser UCU 0.25 0.17 Ala GCC 0.23 0.59 Ser UCC 0.19 0.29 Arg AGG 0.28 0.20 Arg CGG 0.12 0.24 Arg AGA 0.23 0.10 Arg CGA 0.19 0.15 Ser AGU 0.18 0.10 Arg CGU 0.12 0.10 Ser AGC 0.16 0.27 Arg CGC 0.07 0.22 Lys AAG 0.40 0.73 Gin CAG 0.55 0.08 Lys AAA 0.60 0.27 Gin CAA 0.45 0.20 Asn AAU 0.65 0.21 His CAU 0.73 0.22 Asn AAC 0.35 0.79 His CAC 0.27 0.78 Met AUG 1.00 1.00 Leu CUG 0.29 0.49 lie AUA 0.16 0.10 Leu CUA 0.07 0.08 Ile AUU 0.56 0.15 Leu CUU 0.14 0.05 lie AUC 0.28 0.75 Leu cue 0.19 0.27 Thr ACG 0.05 0.15 Pro CCG 0.04 0.14 Thr ACA 0.40 0.15 Pro CCA 0.52 0.22 Thr ACU 0.31 0.12 Pro ecu 0.32 0.19 Thr ACC 0.25 0.58 Pro CCC 0.12 0.45 a Given as fraction of codons for a given amino acid. Data for human C3 are derived from DeBruijn, M.H.L., and G.H. Fey. 1985. Human complement component C3: cDNA coding sequence and derived primary structure. Proc. Natl. Acad. Sci. USA 82:708. TABLE II Amino acid composition of cobra C3 and its α- and /3-chainε TABLE III Sequence homology of cobra C3 with C3 from other εpecieε I-L. CVFl and CVF2: Materialε and Methodε Materialε: Solutionε for RNA iεolation, λgtll cloning, and hybridization probe labeling were obtained from Amersham (Skokie, IL) . In addition, an RNA isolation kit was purchased from Stratagene (La Jolla, CA) . Reagents for cDNA preparation were obtained either from Gibco-BRL (Gaithersburg, MD) , or from Amerεham. Oligo dT-celluloεe waε from Boehringer Mannheim (Indianapolis, IN) , or from Invitrogen (San Diego, CA) . Restriction enzymes were from either Pharmacia, from New England Biolabs (Beverley, MA) , or from Gibco-BRL. Plasmids pUClδ and pUC19 were purchased from Boehringer Mannheim (Indianapolis, IN) , while M13mpl8 and M13mpl9 were purchased from New England Biolabs. DNA modification enzymes were obtained from Pharmacia, New England Biolabs, or Gibco-BRL, and DNA sequencing reagents were obtained from United States Biochemicals (Cleveland, OH) . Reagents required for PCR amplification of venom gland library enεens were obtained from Perkin-Elmer Cetus. Oligonucleotides for screening the libraries were obtained from Clontech (Palo Alto, CA) , or were synthesized, using an Applied Biosyεtems #380 DNA synthesizer. A GeneCleanll kit, containing reagents uεed for isolation of DNA from agarose gels, and for the purification of PCR products, was obtained from BiolOl (La Jolla, CA) . Nitrocellulose and nylon membranes for plaque lifts were obtained from Schliecher and Scheull (Keene, NH) . Rabbit anti-goat IgG (Alkaline Phosphataεe conjugated) waε obtained from Sigma (St. Louis, MO) . [α- 32 P]dATP, [α- 32 P]dCTP, and [α- 35 S]dATP were obtained from Amersham. Methods: RNA Iεolation from Cobra Venom Glandε: Adult cobraε (Naja naja, 1.5-2 eterε in length) were anesthetized with katamine (70 μg/kg i.m.) and with halothane/oxygen by intubation, essentially as described (Vogel, C.-W., and H.J. Muller-Eberhard. 1985. The cobra complement system: I. The alternative pathway of activation, Dev. Comp. Immunol . 9:311). Venom glands were removed and immediately frozen in liquid nitrogen. For RNA preparation, approximately 1 gram of tissue was suspended (while frozen) in 20 ml of a solution of 4 M guanidinium thiocyanate and 1.14 M /S-mercaptoethanol, and the RNA extracted according to the instructions supplied with the Amersham RNA Extraction Kit. This procedure was based on a publiεhed procedure (Han, J.H., C. Stratowa, and W.J. Rutter, 1987. Iεolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning. Biochemistry 26:1617). Poly-A containing RNA was then isolated by chromatography over oligo-dT cellulose, (Jacobson, A. Purification and fractionation of poly(A)* RNA. 1987. In Methods in Enzymology, Vol. 152. S.L. Berger and A.R. Kimmel, edε. Academic Press, Orlando, FL, p. 254) . Whole RNA was also prepared using the Stratagene RNA isolation kit, in which the organs were homogenized in the presence of guanidinium isothiocyanate and β- mercaptoethanol, followed by phenol extraction and isopropanol precipitation (Chomczynski and Sacchi, 1987) . Following extraction, poly A+ RNA was prepared by chromatography over oligo-dT cellulose, aε described above. cDNA Svnthesiε and Cloning: Cobra venom gland cDNA waε synthesized (Krug, M.S., and S.L. Berger. 1987. First-εtrand cDNA synthesis primed with oligo(dT) . In Methods Enzymology. Vol. 152. S.L. Berger and A.R. Kimmel, edε. Academic Press. Orlando, FL, p. 316; Gubler, U. 1987. Second-strand cDNA εyntheεis: mRNA fragments as a primer. In Methods in Enzymology, vol. 152. S.L. Berger and A.R. Kimmel. edε. Academic Preεs, Orlando. FL. p. 330) using the cDNA synthesis kit from Amersham. cDNA was synthesized using both oligo-dT and random hexamers as the primers. cDNA was then prepared for cloning into λgtll (Wu. R. , T. Wu. and A. Ray. 1987. Adaptors, linkers, and methylation. In Methods in Enzymology. vol. 152. S.L. Berger and A.R. Kimmel, eds. Academic Press, Orlando, FL. p. 343) , and the recombinant λ clones were packaged (Hohn, B., and K. Murray. 1977. Packaging recombinant DNA molecules into bacteriophage particles in vitro. Proc. Natl . Acad. Sci . USA 74:3259). E. coli Y1090 (r, m+) waε uεed aε the hoεt for recombinant λgtll. In addition, cDNA waε prepared uεing Superscript (RNase H " ) MMLV Reverεe Transcriptase (Gerard et al, 1989) . In this case, double stranded cDNA was εized on a 1% Agarose gel in TAE buffer. cDNA greater than 4.5 kb was excised from the gel, and the DNA extracted from the agarose using the GeneCleanll kit from BiolOl. Thiε cDNA waε then cloned into the plasmid pSPORT (Chen and Segburg, 1985) , and the recombinant plasmids transformed into E. coli DH5α competent cells. Screening of λgtll Libraries: Libraries were screened (Young, R.A. , and R.W. Davis. 1983. Yeast RNA polymerase II genes: isolation with antibody probes. Science 222:778; Huynh, T.V. , R.A. Young, and R.W. Davis. 1985, Construction and screening cDNA libraries in λgtlO and λgtll. In DNA Cloning vol. 1. A Practical Approach. D.M. Glover, ed. IRL Preεε, Oxford, p 49) . The primary antibody for εcreening the venom gland library waε goat anti-CVF antiεerum. Further plaque purification waε done as described above, using succesεively lower plaque densities. Later screening was done by the hybridization protocol (Wahl, G.M. , and S.L. Berger. 1987. Screening colonies of plaques with radioactive nucleic acid probes. In Methods in Enzymology, vol. 152. S.L. Berger and A.R. Kimmel, eds. Academic Preεs, Orlando, FL, p. 415), using the clones derived from the antibody screening as probes. These probes were labeled with [α- 32 P]dATP or [α- 32 P]dCTP (Feinberg, A.P., and B. Vogelstein. 1983. A technique for radio-labeling DNA restriction endonuclease fragments to high specific activity. Anal . Biochem. 132:6). pSPORT libraries were screened using other cDNA clones as a probe. Subcloning and DNA Sequence Analysis: Clones containing CVF inserts were grown up on agarose plates and their DNA prepared as described (Maniatis, T. , E.F. Fritεch, and J. Sambrook. 1982. Molecular Cloning - A Laboratory Manual . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Inserts were prepared by EcoRI digestion, followed by agarose gel electrophoresis. In some cases, λgtll inserts were isolated using the polymerase chain reaction (PCR) on a Savant Model TC4 thermal cycler. In this case, λgtll amplimers from Clontech were used aε primers, and the inserts were amplified using the protocol supplied with the amplimers. Thiε conεiεted of 30 cycleε with a 15 εec. denaturing stop at 94°C, 15 sec. of annealing at 58°C and a 1 minute extension at 72°C in a 50 μl reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl 2 , 1 mM in each deoxynucleoside triphosphate, 1 μM in each primer, 1.25 U of A plitaq® polymerase, and approximately 100 ng DNA to be amplified. Following amplification, the insertε were purified with the BiolOl GeneCleanil kit, the DNA digested with EcoRI, and electrophoreεed through an agaroεe gel. In all cases, fragments were eluted from the gel using the IBI Electroeluter (Model UEA) or by using the GeneCleanil kit from BiolOl. The DNA inserts were then ligated into pUC18 (Yanisch-Perron, C,J. Vieira, and J. Meεεing, 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequenceε of the M13 mpl8 and pUC19 vectorε. Gene 33:103) and transformed into E. coli JM105 to facilitate the production of large quantities of the insert. Subfragmentε of the CVF inεerts were subcloned into M13mpl8 or mpl9 (Yanisch-Perron, C.,J. Vieira, and J. Mesεing, 1985. Improved M13 phage cloning vectors and host strainε: nucleotide εequenceε of the M13 mpl8 and pUC19 vectors. Gene 33:103) for sequence analysis. Sequencing was performed using the dideoxy-chain termination technique (Sanger, F.,S. Nicklen, and A.R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl . Acad. Sci . USA 74:5463). The Sequenase Version 2.0 sequencing kit from U.S. Biochemicals (Tabor, S., and CC Richardson. 1989. Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA Polymerase 1. Proc. Natl . Acad. Sci . USA 86:4076) was used aε the εource of enzymes, chemicals and primers for sequencing. The DNA sequence was assembled and analyzed using the group of sequence analysis programs written by the Genetics Computer Group of the Wisconεin Biotechnology Center (Devereux, J.R. , P. Haeberli, and O.A. Smithieε. 1984, Comprehenεive εet of sequence analysiε programs for the VAX. Nucleic Acids Res . 12:387) . Reεults Screening of the cobra venom gland library. Poly-A+ RNA from cobra venom glands was used for the preparation of cDNA that waε cloned into the EcoRI εite of λgtll. Librarieε were prepared from cDNA that had been primed with oligo-dT and with random hexamers. Each library contained at least 5x10° clones. Initially, 5x10 s clones from the random primed library were screened using CVF specific antisera to detect clones producing CVF containing fusion proteins. In the first round of screening, a single positive clone (CVF5, 1.1 kb. Figure 13) was isolated. Sequence analysis of this clone revealed that it contained a single open reading frame of 639 nucleotides comprising the C-terminal 213 amino acid residues of the /S-chain, and an 3'-untranslated region of 502 nucleotides. Thiε represented approximately 12% of the mature protein. To obtain clones representing the rest of the CVF message, several strategies were used. First, the oligo-dT primed library was screened by uεing hybridization, using CVF5 as a probe. This resulted in the isolation of several clones, one of which (CVF18, 2.6 kb) was used for further sequence analysis. CVF18 contains the 3'-end of the CVF message, with an open reading frame of 1002 nucleotides, and 3' untranslated region of 994 nucleotideε. Hybridization screening of an oligo-dT primed venom gland library yielded the clone CVF106 (3.5 kb) . Clones containing the 5' end of the CVF cDNA were isolated by screening the random primed λgtll venom gland library by hybridization, using upstream restriction fragments of sequenced DNA as probes. By this means, two additional clones, CVF65 (3.4 kb) and CVF72 (2.0 kb) were isolated for sequencing. Figure 13 shows the placement of the clones used for sequencing on the CVFl mRNA. Structure of the Cobra Venom Factor cDNA. The CVFl cDNA is 5924 nucleotides in length. It contains a εingle open reading frame of 4926 nucleotides, coding for a pre- pro-protein of 1642 amino acid residues (Figure 14) . The cDNA has a 5' untranslated region of 3 nucleotides, and a 3' untranslated region of 994 nucleotides. The start of the poly-A tail has not been found. The coded pre-pro-protein has a signal sequence of 22 residues with a core rich in hydrophobic amino acids. The signal εequence iε followed by the 627 amino acid α-chain. The α-chain haε three glycoεylation εiteε at residues 131, 136, and 187. Immediately following the C-terminus of the α-chain, there are 4 arginine reεidueε, and a 68 amino acid peptide reεembling the C3a anaphylatoxin. There iε a εingle glycosylation site at poεition 640, though thiε site is not present in the mature protein. The 7-chain begins at position 710, and extends for approximately 300 amino acid residueε. The poεition of the C-terminuε of the 7-chain iε unknown, and is apparently heterogeneous. The 7-chain contains no glycoεylation εites. The /S-chain of CVF begins at position 1242, and extends for 378 residues to the end of the open reading frame. The /S-chain contains a single glycosylation site at position 1324. The G+C composition of the open reading frame for CVFl is 43.5% (for the whole cDNA: 42.4%). This is approximately the same as cobra C3, though lower than that for sequenced mammalian C3s. Homology to cobra and other C3 proteins. The CVFl sequence was compared to C3 sequences from cobra, human, and mouse. CVFl showε a high degree of homology to cobra C3 at both the nucleic acid and protein level. At the protein level, CVFl iε nearly 85% identical to cobra C3 (greater than 91% εimilar if conservative replacements are allowed) , while the nucleic acid εequenceε of the two messages are greater than 93% identical. CVFl alεo εhowε a high, though lesser degree of homology to human and mouse C3 sequences. For example, the protein sequence of CVF is nearly 50% identical to that of human C3 (more than 69% similar if conservative replacements are allowed) , while the nucleic acid sequence is nearly 57% identical. Comparing the CVF sequence to that of mouse C3, we find that the protein sequences are more than 51% identical (70% similar) , and the nucleic acid sequences are nearly 58% identical. Dotplot comparisons of the CVFl protein sequence with that of cobra and human C3 show that the homologies are spread throughout the molecule. The homology between CVFl and mammalian C3s is markedly higher than the average at certain ligand binding siteε. For example, at the Factor B binding site, near the Nterminus of the 7-chain, CVF is 90% similar (though it is only 30-40% identical) to the homologous regions of other sequenced C3s (Figure 15) . The homology is also quite high at the properdin binding site (Figure 16) , where greater than 53% of the amino acid residues are identical, and about 80% of the amino acids are similar. Interestingly, some sites are well conserved, even though they are not present in the mature protein. The best example of this is the sequence around the internal thioester site, where approximately 70% of the sequence is identical, and 80% is similar (Figure 17) . At the Factor H binding site, which is also not present in the mature protein, the homology is not noticeably greater than in the rest of the protein (Figure 18) . However, there is a stretch of 9 amino acid residues in the second portion of the discontinuous Factor H binding site (1192-1200 in CVFl) that is strictly maintained in all of the sequences examined, including rat and rabbit (data not shown) , implying conservation of at least part of the Factor H binding site. Additional clones encoding a distinct CVF were also isolated. The protein encoded by this gene is referred to as CVF2, and a partial sequence for CVF2 is shown in Figure 22. The clones containing the DNA of CVF2 were obtained in the same series of experimentε which gave the DNA for CVFl. Full length cloneε of C3 or CVFl or CVF2 may be obtained by preparing another library containing full length cloneε or preparing the cloneε by ligating together the preεent cloneε uεed to εequence C3, CVFl, or CVF2. The method choεen for thiε iε to uεe PCR to amplify εectionε of cDNA, and to ligate the fragmentε together to give a full length clone. Specifically, the CVFl cDNA εequence haε been εearched for unique reεtriction sites at positionε approximately one-third and two-thirdε of the way through the molecule, and the εiteε surrounding these sites used aε primerε for the PCR reaction. In addition, a 3' primer haε been designed, containing a εtring of T reεidueε and εequenceε with a unique NotI εite. A 5' primer also has been designed that containε unique Sail and Mlul sites. After amplification of the 3 fragmentε, each fragment will be cut with the unique enzyme, and the fragmentε ligated together to form the 6 kb ligation product. Thiε product may be cut with Sail and NotI, ligated into the vector pSport (Gibco/BRL) and tranεformed into the E. coli strain DH5α. pSport allows tranεcription of the inserted gene (in either orientation) , using either T3 or T7 RNA polymeraεe. In addition, by cutting with NotI and SAII, the full length CVF clone may be removed from pSport aε a caεεette that may be clonable into other expreεεion vectorε for expreεεion in bacterial or eukaryotic hosts. a) Preparation of full length clones of CVF. To prepare full length clones of CVF, approximately 2 kb fragments of CVF are prepared using PCR amplification. Specifically, a 2 kb 5' fragment is prepared using a 5'oligo (GCGTCGACCCACGCGTCCGCCATGGAGAGGATGGC) that represents the 5' 17 nucleotides of the sequenced CVFl cDNA, along with 18 nucleotides containing unique Sail and Mlul siteε. The other oligo (CTGCGACGCCTCCGATTTGCAGGC) iε complementary to a portion of the sequence 1945 nucleotideε from the 5' end, that containε a unique Hgal εite. The template iε the λ clone, CVF72. The amplification takeε place in a Savant TC49 thermal cycler, in a 100 μl reaction that is 10 mM Tris -HCI (pH 8.3), 2 mM MgCl 2 , 50 mM KCl, 200 μM in each dNTP, 1 μM in each oligonucleotide, 0.1 μg template DNA, and containing 2.5 units of Amplitaq® polymerase. Amplification consists of one cycle of; 94°C for 120 sec, 45 β C for 45 sec, and 72 β C for 90 sec, followed by 34 cycles of: 94 β C for 45 sec, 45°C for 45 sec, and 72°C for 90 sec. After the final cycle the reactionε are incubated for a further 10 min at 72°C, and εtored at 4°C until ready for analyεiε. The other fragmentε are prepared aε above, but with different templates and oligonucleotides. For the central fragment, the 5' oligo is complementary to the 3' oligo for the 5' fragment, while the 3' oligo (GCATTTTCATAATTAATTCTGTACC) iε complementary to the CVFl εequence at poεition 3878, covering a unique Aεel εite. The template for the central fragment is a ligation of a 2.5 kb EcoRI fragment from CVF65, and a 1.5 kb EcoRI fragment from pCVF106. The 3' fragment is prepared using, as a 5' oligo, an oligonucleotide complementary to the 3' oligo used for synthesizing the central fragment, while the 3' oligo (GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTT) consiεtε Of a unique NotI εite followed by a string of 12 T residues. The template for the 3' fragment is pCVF106. Following preparation of the three fragments, they are purified using the GeneClean kit (BiolOl). The 3' and central fragmentε are both cut with Asel, and ligated together in a 20 μl reaction containing 50 mM Tris-HCl (pH 7.8), 10 mM MgCl 2 , 10 mM 2-mercaptoethanol, and 1 mM ATP, with each fragment at a concentration of approximately 25 μg/ml. The ligations are performed for 16 hrs at 16 β C, and the ligase is heat inactivated by incubation at 65°C for 15 min., followed by digestion of the ligation mixture with Hgal for 3 hrs at 37°C. The productε are εeparated on a 0.8% agarose gel, and the 4 kb product is isolated from the gel (uεing the GeneClean kit), and ligated to the 5' fragment (that had been Hgal digeεted and purified with the GeneClean kit) aε described above. The ligation product iε cut with Sail and NotI, ligated into NotI and Sail cut pSPORT (Gibco/BRL) , and tranεformed into competent DH5α cells. Proper recombinant clones are identified by reεtriction mapping and Southern blotting. To examine the tranεlation products of the putative fully length CVF clones, RNA iε prepared in vitro, uεing transcription by T7 RNA polymerase, followed by translation of the RNA products in a Rabbit Reticulocyte translation system. First, the insert iε transcribed in a 20 μl reaction containing 50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 6 mM MgCl 2 , 2 mM Spermidine, 10 mM DTT, 0.5 mM in each ribonucleotide triphosphate, 0.5 μg CVF containing plasmid, and 10 units of T7 polymerase. The reaction is incubated for 1 hour at 37°c, and the reaction ended by phenol extraction and EtOH precipitation of the RNA. The RNA iε resuspended in 20 μl of RNaεe-free H 2 0, AND 10 μl will be tranεlated in a 100 μl reaction containing 21 mM Hepeε (pH 7.4), 0.5 nM spermidine, 8.5 mM Creatine phosphate, 30 μM in each of 20 amino acids, 2 mM DTT, 80 mM KCl, and 75 μl rabbit reticulocyte lyεate (Promega, Madiεon WI) . The reaction are incubated for 2 hr at 30°C, and loaded on a 7.5% polyacrylamide gel run in the preεence of SDS. The productε are analyzed by WEεtern blotting, followed by detection with goat anti-CVF antiεera. Any cloneε producing immunoreactive full length protein products are partially sequenced, using the dideoxy-chain termination technique, and any cloneε without major alterations in sequence are used for in vitro mutagenesis experiments. b) Expreεεion of CVF cloneε in eukaryotic cells. Transient expression studies of CVF are done by transfecting CHO of NIH 3T3 cells with CVF εequenceε cloned into the mammalian expreεεion vector pMT2, containing the SV40 origin of replication and early gene enhancer, the adenovirus major late promoter fuεed to the adenovirus tripartite leader, a hybrid intron splice site, and the SV40 polyadenylation εignal. The CVF cDNA iε ligated into the unique EcoRI εite that iε between the intron and the polyA addition site, and recombinant plasmidε are transformed into E. coli DH5α. Recombinant clones are checked for the orientation of the insert by restriction analysiε. Plaεmids containing the CVF insert in the proper orientation are isolated and purified by two rounds of isopycnic CsCl 2 gradient centrifugation, and transformed into COS cells by calcium phosphate mediated transfection as described above. The transformed cells are then grown for 24 hrε, and both the cells and the media are assayed for the CVF production by Western analysis as described above. For production of larger quantities of CVF, the baculovirus expression system is uεed. In thiε εyεtem, a plasmid containing the gene to be expressed is co- transfected into Spodopera frugiperda (Sf9) cells, along with the wild type Autographica californica nuclear polyhedrosis virus (AcNPV) . Following transfection, a 0.1 to 5% of the wild type viruses acquire the gene to be expresεed by homologouε recombination. To clone CVF cDNA into baculovirus, the CVF casεette iε removed from the original CVF clone by cleavage with Sail and notl, and cloned into the Nhel site of the baculovirus cloning vector pJV(Nhel) . This vector, in addition to having the polyhedron promoter and poly-A addition site, also contains a copy of the /3-galactosidase gene attached to the PlO promoter (another very late promoter) . Recombinants are screened by restriction analysiε and DNA εequencing to find cloneε with the CVF cDNA in the right orientation. The recombinant pJV(Nhel) iε then co-tranεfected with wild type AcNPV into Sf9 cellε, using the calcium phosphate precipitation technique. After transfection, the cells are overlayed with 10 ml of 1% agarose (for a 100 x 15 mm culture plate) diluted with Graceε media (Life Technologieε, Inc., Gaitherεburg, MD) , and are incubated at 27 β C After 3 dayε, the plateε are overlaid with the εame agar, containing 150μg/ml Bluo-gal (Life Technologieε, Inc., Gaitherεburg, MD) . After a further 6 hr incubation, cellε infected with recombinant viruεeε produce blue plaqueε from the digestion of the Bluo-gal by β- galactoεidaεe. These plaqueε are picked, and the phage allowed to elute into Graceε medium overnight. Thiε plaque purification technique iε repeated until all plaqueε are blue, indicating that the virus stock is pure. To express CVF in ACNPV infected Sf9 cells, cells are seeded into 25 cm 2 flasks at a density of 3 X 10 s cells/flask, and allowed to attach for one hour. The 1 ml of a suspenεion of recombinant virus is added so that the multiplicity of infection is between 5 and 10, and the infection is allowed to proceed for 1 hr. Then, the virus suspension is replaced with freεh medium, and the cellε allowed to grow for 2 to 4 dayε. The cellε are then harvested, and the cellε and medium checked for the presence of CVF by SDS aery1amide gel electrophoresis, followed by Western blotting. Once produced, the recombinant CVF is purified by standard methods, using reaction with anti-CVF antiεera aε an assay for the presence of the protein. Larger quantities of CVF are produced by scaling up the growth of infected cells. It should be understood that cobra C3, CVFl, or CVF2 may be prepared by expressing a εingle DNA εequence which encodes, e.g., pre-pro-cobra C3, pro-cobra C3, pre-pro- CVFl, pro-CVFl, pre-pro-CVF2, or pro-CVF2, with any post tranεlational processing being carried out as described above. Alternatively, cobra C3, CVFl and CVF2 may be prepared by expresεing a plurality of DNA εequenceε which each encodes a fragment of the whole molecule. For example C3 may be prepared by separately preparing the α-chain of C3 and the /3-chain of C3, and then incubating the α- and β- chains together. The DNA sequences encoding the fragments may be contained the same or different plasmids and in the same or different transformed hosts. Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be underεtood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims: Claims 1. A biologically pure segment of DNA, comprising a sequence of DNA which encodes a polypeptide having the amino acid sequence: (a from position about -22 to position about 1629 in Figure 2; (b from position about 1 to position about 1629 in Figure 2; (c from poεition about 638 to poεition about 1629 in Figure 2; (d from poεition about 1 to poεition about 633 in Figure 2; (e from poεition about 717 to poεition aobut 1629 in Figure 2; or (f from poεition about 638 to poεition aobut 716 in Figure 2. 2. The DNA of Claim 1, comprising the DNA εequence: (a ) from poεition about 1 to poεition about 5211 in Figure 2; (b ) from poεition about 9 to poεition about 4961 in Figure 2; (c ) from poεition about 75 to poεition about 4961 in Figure 2; (d ) from poεition about 1986 to poεition about 4961 in Figure 2; (e ) from poεition about 75 to poεition about 1973 in Figure 2; (f ) from positon aobut 2223 to position about 4961 in Figure 2; or (g ) from position about 1986 to poεition about 2222 in Figure 2. 3. A biologically pure segment of DNA, compriεing a εequence of DNA which encodeε a polypeptide having the amino acid εequence: (g) from poεition about -22 to poεition about 1620 in Figure 14; (h) from position about 1 to position about 1620 in Figure 14; (i) from position about 1 to position about 627 in Figure 14; (j) from position about 711 to position about 1010 in Figure 14; (k) from poεition about 1242 to position about 1620 in Figure 14. (1) from position about 1 to position about 1010 in Figure 14; or (m) from position about 711 to position about 1620 in Figure 14. 4. The DNA of Claim 3, comprising the DNA sequence: (h') from position about 1 to position about 5924 in Figure 14; (i') from position about 4 to position about 4929 in Figure 14; (j') from position about 70 to position about 4929 in Figure 14; (k') from position about 70 to position about 951 in Figure 14; (1') from position about 2200 to position about 3299 in Figure 14; ( ') from position about 3793 to position about 4929 in Figure 14; (n') from position about 70 to position about 3299 in Figure 14; or (o') from position about 2200 to position about 4929 in Figure 14. 5. A biologically pure segment of DNA, comprising a sequence of DNA which encodes a polypeptide having the amino acid sequence: (n) in which the carboxy-terminus of the amino acid εequence of from position about -22 to position about 299 in Figure 14 is bonded to the amino- terminus of the amino acid sequence of from position about 1 to poεition about 1333 in Figure 22; (o) in which the carboxy-terminus of the amino acid sequence of from position about 1 to position about 299 in Figure 14 iε bonded to the amino- terminus of the amino acid sequence of from position about 1 to position about 1333 in Figure 22; (p) in which the carboxy-terminus of the amino acid sequence of from position about 1 to position about 299 in Figure 14 iε bonded to the amino- terminus of the amino acid sequence of from position about 1 to poεition about 332 in Figure 22; (q) from position about 416 to position about 715 in Figure 22; (r) from position about 947 to position about 1333 in Figure 22; (s) in which the carboxy-terminuε of the amino acid εequence of from position about 1 to position about 299 in Figure 14 is bonded to the amino- terminus of the amino acid sequence of from position about 1 to position about 715 in Figure 22; or (t) from position about 416 to position about 1333 in Figure 22. 6. The DNA of Claim 5, compriεing the DNA εequence: (p') in which the 3'-terminuε of the DNA εequence from poεition about 1 to poεition about 964 in Figure 14 iε bonded to the 5'-terminuε of the DNA sequence from position about 1 to poεition about 4138 in Figure 22; (q') in which the 3'-terminuε of the DNA εequence from position about 4 to position about 964 in Figure 14 iε bonded to the 5'-terminuε of the DNA εequence from position about 1 to position about 4001 in Figure 22; (r') in which the 3'-terminuε of the DNA εequence from poεition about 70 to position about 964 in Figure 14 is bonded to the 5'-terminus of the DNA sequence from position about 1 to position about 4001 in Figure 22; (s') in which the 3'-terminus of the DNA sequence from position about 70 to poεition about 964 in Figure 14 iε bonded to the 5'-terminus of the DNA sequence from position about 1 to position about 998 in Figure 22; (f) from position about 1248 to position about 2147 in Figure 22; (u') from position about 2841 to position about 4001 in Figure 22; (v') in which the 3'-terminus of the DNA sequence from position about 70 to poεition about 964 in Figure 14 iε bonded to the 5'-terminuε of the DNA sequence from position about 1 to position about 2147 in Figure 22; or (W) from position about 1248 to position about 4001 in Figure 22. 7. A plasmid, comprising the DNA of Claim 1. 8. A plasmid, comprising the DNA of Claim 2. 9. A plasmid, comprising the DNA of Claim 3. 10. A plasmid, comprising the DNA of Claim 4. 11. A plasmid, comprising the DNA of Claim 5. 12. A plasmid, comprising the DNA of Claim 6. 13. A transformed host, comprising the plasmid of Claim 7. 14. A transformed host, comprising the plaεmid of Claim 8. 15. A transformed host, comprising the plasmid of Claim 9. 16. A transformed host, comprising the plasmid of Claim 10. 17. A transformed hoεt, comprising the plasmid of Claim 11. 18. A transformed host, comprising the plaεmid of Claim 12. 19. A method for producing a polypeptide, compriεing culturing the transformed hoεt of Claim 13. 20. A method for producing a polypeptide, compriεing culturing the transformed host of Claim 14. 21. A method for producing a polypeptide, comprising culturing the transformed host of Claim 15. 22. A method for producing a polypeptide, comprising culturing the transformed host of Claim 16. 23. A method for producing a polypeptide, compriεing culturing the transformed host of Claim 17. 24. A method for producing a polypeptide, compriεing culturing the transformed host of Claim 18.

Inventors: BREDEHORST REINHARD, FRITZINGER DAVID C, VOGEL CARL-WILHELM, BREDEHORST, REINHARD, FRITZINGER, DAVID, C., VOGEL, CARL-WILHELM
Applicants: UNIV GEORGETOWN, GEORGETOWN UNIVERSITY
Application year: 1994
Main classification: C12N15
IPC codes: C07K14, C07K19, C12N15